1. Field of the Invention
The present invention relates to antimicrobial agents. More specifically, the present invention relates to a series of novel positively-charged porphyrins having at least two and up to four positive charges situated in peripheral substituents of the tetrapyrrolic macrocycle and at least one hydrophobic tail originating from one of such charged sites or two adjacent charged sites which are active as photodynamic agents in killing both Gram-positive and Gram-negative bacteria.
2. Description of Related Art
The resistance to antibiotics developed by an increasing number of microorganisms is recognized to be a worldwide health problem. Tunger et al., Int. J. Antimicrob. Agents, 15:131-135 (2000); Jorgensen and Ferraro, Clin. Infect. Dis. 30:799-808 (2000). Historically, the discovery and development of new antibiotics has been a slow process plagued with uncertainty, disappointment, and only relatively rare successes. Even after discovery, prospective drug candidates must undergo rigorous testing to assure their safety and efficacy. Because of this, the highly reported advent of drug-resistant microorganisms has caused alarm among medical professionals and the public, with many left wondering whether novel antibiotics can be developed quickly enough to forestall possible problems.
As a result of this, researchers have begun to explore the possibilities of developing nontraditional antibiotic approaches for killing microorganisms. The goals of such development efforts include not only controlling antibiotic-untreatable infections, but in addition, limiting the development of additional antibiotic-resistant microbe strains by selecting a killing mechanism which does not involve the target""s genetic material, or which is not otherwise mutagenic. This acts to prevent, at least in part, selection for or creation of strains potentially resistant to the action of the killing agent.
One such method being evaluated is the treatment of microbial infections by photodynamic therapy (xe2x80x9cPDTxe2x80x9d). This appears to be a valuable alternative method of eradicating bacteria, in part, because it appears to utilize a mechanism that is different from that typical of most antibiotics. Generally, PDT is based on the use of a photosensitizing molecule that, once activated by light, generates reactive oxygen species (xe2x80x9cROSxe2x80x9d) that are toxic to a large variety of prokaryotic and eukaryotic cells including bacteria, mycoplasma, and yeasts. Malik et al., J. Photochem. Photobiol. B: Biol., 5:281-293 (1990); Bertolini et al., Microbios, 71:33-46 (1992).
One important feature of this approach is that the photosensitizing activity of many photodynamic agents is not impaired by bacterial resistance to antibiotics. Instead, it largely depends on the chemical structure of the photosensitizing agents themselves. Malik et al, J. Photochem. Photobiol. B: Biol., 14:262-266 (1992). Various types of known neutral and anionic photosensitizers, for example, exhibit a pronounced phototoxic activity against Gram-positive bacteria while exhibiting no appreciable cytotoxic activity against Gram-negative bacteria unless the permeability of the outer membrane of the Gram-negative bacteria is altered by treatment with EDTA or polycations. Bertolini et al., FEMS Microbiol. Lett., 71:149-156 (1990); Nitzan et al., Photochem. Photobiol., 55:89-97 (1992). Without being limited to any one theory, it appears, in light of current research, that the more complex and thicker cellular envelope of Gram-negative bacteria (as compared to that of Gram-positive bacteria) may prevent the efficient binding of these photosensitizer molecules. In addition, the envelope may simply intercept and deactivate the cytotoxic reactive oxygen species generated by the photosensitizer molecules before fatal damage can be inflicted. Ehrenberg et al., Photochem. Photobiol., 41:429-435 (1985); and Valduga et al., Photochem. Photobiol. B: Biol., 21:81-86 (1993).
In contrast, positively charged photosensitizers, including porphyrins and phthalocyanines, promote the efficient inactivation of Gram-negative bacteria without the need of modifying the natural structure of the cellular envelope. Merchat et al., J. Photochem. Photobiol. B: Biol., 32:158-163 (1996); and Minnock et al., J. Photochem. Photobiol. B: Biol., 32:159-164 (1996). Again, without being limited to any one theory, it appears that the positive charge favors the binding of the photosensitizer molecule at critical cellular sites which, once damaged by exposure to light, cause the loss of cell viability. Merchat et al., J. Photochem. Photobiol. B: Biol., 35:149-157 (1996).
One of the families of positively-charged photosensitizers currently being investigated is based on the porphyrin molecule. Porphyrins are macrocyclic molecular compounds with a ring-shaped tetrapyrrolic core. As such, porphyrins are commonly found in their dianionic form coordinated to a metal ion. The unique properties of the tetrapyrrolic core have made porphyrins central in many biological systems that play a vital role in many life processes. Several compounds which are critically important for essential biological processes, such as chlorophyll and heme, are derived from the coordination of a metal ion with a porphyrin nucleus. H. R. Mahler and E. H. Cordes, Biological Chemistry, 2d ed. 418, 1966.
Porphyrins are generally derived from the parent tetrapyrrole porphin by replacing hydrogens at one or more of the positions 1 and 8 as well as at one or more of the meso-(pyrrole bridging) carbon atoms with side chains such as, for example, methyls, ethyls, vinyls, propionic acids, or aromatic groups. Id. Porphyrins are often classified based on the side chains they contain. Hydrogenation of one or two pyrrole moieties generates the corresponding chlorophyll, and, respectively, bacteriochlorophyll derivatives.
As briefly noted above, porphyrins are able to form metal chelates with a large variety of metal ions, including: cobalt, copper, iron, magnesium, nickel, silver, and zinc. Id. at 419. Heme is an iron chelate of a porphyrin, while chlorophyll and bacteriochlorophyll are magnesium chelates. Porphyrins such as these are generally synthesized from the precursors glycine and succinyl CoA. See L. Stryer, Biochemistry, 2d ed. 504-507 (1981).
Present porphyrins and techniques for their use require that, in order to act as antimicrobial agents in the treatment of bacterial and other microbial infections, or even for use in the photosterilization of water, they must be employed in concentrations of at least 10 micromolar. They must then be irradiated for as long as about 30 minutes. See id.
One porphyrin-based cationic photosensitizer shown to be effective in killing both Gram-positive and Gram-negative bacteria, including the ability to efficiently inactivate Escherichia coli, is cationic meso-tetra(N-methyl-4-pyridyl)porphine, or xe2x80x9cT4MPyPxe2x80x9d. See Merchat, et al., J. Photochem. and Photobiol. B: Biol. 32:153-157, (1996); Merchat, et al., J. Photochem. and Photobiol B: Biol., 35:149-157, (1996); Okuno, Synthesis, July 1980, 537, and Valduga et al., Biochem. Biophys. Res. Comm., 256:84-88 (1999). Without being limited to any one theory, it appears that the phototoxic activity of this porphyrin is mediated by the impairment of enzymatic and transport functions of both the outer and cytoplasmic membrane. DNA was found not to be a primary target of T4MPyP photosensitization.
It has further been well established that the hydro- or lipo-philicity of a photosensitizer strongly affects the binding of the photosensitizer to a target cell, and as a consequence, its cytotoxic activity. Merchat et al., J. Photochem. Photobiol. B: Biol., 35:149-157 (1996).
Currently known porphyrin photosensitizers, current methods of their synthesis, and known techniques for their use are inadequate for many intended applications. This is true in part due to the need for high concentrations of reagent and the requirement of extended irradiation periods. These factors render the methods burdensome and inconvenient. In addition, such conditions are not suitable for many medical and/or industrial applications.
It is thus seen that there is a need for novel photosensitizing antimicrobial agents for medical or other applications. It would be an improvement in the art to provide an antimicrobial agent that utilizes a pathway for inactivating or killing an organism which is non-mutagenic. It would be a further improvement in the art to provide alternate hydrophobic cationic porphyrin photosensitizers that are suitable for use against both Gram-positive and Gram-negative bacteria, as well as other microbes. Finally, it would be an additional improvement in the art to provide such a photosensitizer that is capable of functioning effectively at lower concentrations and over shorter periods than those currently known and taught in the art.
The compositions of matter of the present invention, the methods of their synthesis, and the associated methods for their use have been developed in response to the present state of the art. In particular, these aspects of the invention have been developed in response to the problems and needs in the art that have not yet been fully solved by currently available positively-charged porphyrins, porphyrin synthetic methods, and methods for the use of porphyrins to kill microbes including Gram-positive and Gram-negative bacteria. Thus, it is an overall objective of the present invention to provide positively-charged photodynamic porphyrins and methods for their use as photodynamic antimicrobial agents which perform better than those in the art.
To achieve the foregoing objects, and in accordance with the invention as embodied and broadly described herein in the preferred embodiment, a group of novel positively charged porphyrins is provided. Many of these novel porphyrins have up to four positive charges. In addition, these novel porphyrins also include at least one hydrophobic tail consisting of at least one hydrocarbon chain of between 6 and 22 carbons (inclusive) in length.
In one embodiment of the invention of the instant application, the photodynamic antimicrobial agent comprises a porphyrin having four quaternized nitrogens. In this form of the invention, the porphyrin further comprises a hydrocarbon tail beginning at one of the quaternized nitrogens. In some instances, the porphyrin is a light-absorbing reduced porphyrin. In some instances, the reduced porphyrin is selected from the group consisting of chlorin and bacteriochlorin. In other instances, the porphyrin or reduced porphyrin is a metal chelated porphyrin. In these instances, metals whose porphyrin chelates are useful as photodynamic antimicrobial agents include Mg (magnesium), Sc (scandium), Zn (zinc), Al (aluminum), In (indium), Tl (thallium), Si (silicon), Ge (germanium), Sn (tin), Pd (palladium), and Pt (platinum).
In other embodiments of the invention, the hydrocarbon tail is chosen from the group of straight chain alkyls, straight chain alkenes, branched alkyl chains, branched alkenyl chains, aromatics, mixed alkyl-aromatic species, and mixed alkenyl-aromatic species. In certain of these, the hydrocarbon tail contains between 6 and 22 carbons (inclusive). In others, the hydrocarbon tail contains between 10 and 18 carbons (inclusive). In some specific embodiments, the hydrocarbon tail comprises a benzyl group. In others, the hydrocarbon tail comprises a six-carbon chain. In yet others, the hydrocarbon tail comprises a ten-carbon chain. In other embodiments, the hydrocarbon tail comprises a fourteen-carbon chain. In still others, the hydrocarbon tail comprises an eighteen-carbon chain.
In other embodiments, the invention of the instant application is a photodynamic antimicrobial agent comprising a porphyrin having four quaternized nitrogens, and further comprising a hydrocarbon tail made up of between 6 and 22 carbons (inclusive), beginning at one of the quaternized nitrogens. In some of these embodiments, the hydrocarbon tail contains between 6 and 18 carbons. In others, the porphyrin is a light absorbing reduced porphyrin. In these, the reduced porphyrin may be selected from the group consisting of chlorin and bacteriochlorin. As above, the hydrocarbon tail is chosen from the group of straight chain alkyls, straight chain alkenes, branched alkyl chains, branched alkenyl chains, aromatics, mixed alkyl-aromatic species, and mixed alkenyl-aromatic species. In yet other embodiments, the porphyrin or reduced porphyrin is a metal chelated porphyrin. In these instances, metals whose porphyrin chelates are useful as photodynamic antimicrobial agents include Mg (magnesium), Sc (scandium), Zn (zinc), Al (aluminum), In (indium), Tl (thallium), Si (silicon), Ge (germanium), Sn (tin), Pd (palladium), and Pt (platinum).
In specific versions of the photodynamic antimicrobial agent of the instant invention, the hydrocarbon tail comprises a benzyl group. In others, the hydrocarbon tail comprises a six-carbon chain. In still others, the hydrocarbon tail comprises a ten-carbon chain. In others, the hydrocarbon tail comprises a fourteen-carbon chain. In still others, the hydrocarbon tail comprises an eighteen-carbon chain.
In a preferred embodiment, the invention comprises a series of derivatives of meso-tetra-(N-methyl-pyridyl)porphyrins, having four quaternized nitrogens, where one N-methyl group is replaced by a hydrocarbon tail ranging from C1 to C22. In some embodiments, the hydrocarbon tail contains between 6 and 22 carbons. In others, the hydrocarbon tail contains between 6 and 18 carbons.
In some of these meso-tetra-(N-methyl-pyridyl)porphyrin derivative embodiments, the porphyrin is a light absorbing reduced porphyrin. In some of these, the reduced porphyrin is selected from the group consisting of chlorin and bacteriochlorin. In others, the porphyrin or reduced porphyrin is a metal chelated porphyrin. In these instances, metals whose porphyrin chelates are useful as photodynamic antimicrobial agents include Mg (magnesium), Sc (scandium), Zn (zinc), Al (aluminum), In (indium), Tl (thallium), Si (silicon), Ge (germanium), Sn (tin), Pd (palladium), and Pt (platinum).
Further, in some of these meso-tetra-(N-methyl-pyridyl)porphyrin derivative embodiments, the hydrocarbon tail is chosen from the group of straight chain alkyls, straight chain alkenes, branched alkyl chains, branched alkenyl chains, aromatics, mixed alkyl-aromatic species, and mixed alkenyl-aromatic species. In some of these, the hydrocarbon tail comprises a benzyl group. In others, the hydrocarbon tail comprises a six-carbon chain. In others, the hydrocarbon tail comprises a ten-carbon chain. In still others, the hydrocarbon tail comprises a fourteen-carbon chain. In still others, the hydrocarbon tail comprises an eighteen-carbon chain.
In other embodiments of the invention, the invention comprises a method of synthesizing porphyrins with four symmetrically distributed positive charges on four quaternized nitrogens and a single hydrophobic hydrocarbon tail. An embodiment of this method of the invention comprises the steps of: providing a porphyrin, quaternizing a first nitrogen of the porphyrin by reacting it with a sub-stoichiometric quantity of a halide salt of the desired hydrophobic hydrocarbon tail, separating the mono-quaternized porphyrins from the non-mono-quaternized porphyrins, quaternizing the remaining nitrogens of the porphyrin using a methylating agent, and separating the resulting porphyrin with four symmetrically distributed positive charges and a single hydrophobic hydrocarbon tail from the reaction mixture. In some embodiments of the invention, either or both of the steps of separating the mono-quaternized porphyrins from the non-mono-quaternized porphyrins and separating the resulting porphyrin with four symmetrically distributed positive charges and a single hydrophobic tail are abridged, simplified, or even omitted when appropriate for a given application.
The halide salt may be selected from the group consisting of straight chain alkyls, straight chain alkenes, branched alkyls, branched alkenyls, aromatics, mixed alkyl-aromatics, and mixed alkenyl-aromatic halide salts. Additionally, the methylating agent may be methyl-p-toluene sulfomate, methyl iodide, dimethyl sulfate, methyl fluorosulfonate, and other suitable methylating agents. The step of separating the mono-quaternized porphyrin from the non-mono-quaternized porphyrins in the method of the instant invention may be accomplished utilizing solubility differences. Further, the step of separating the resulting porphyrin with four symmetrically distributed positive charges and a single hydrophobic tail may be accomplished using chromatography. This step may be simplified or omitted when the intended application of the porphyrins warrants, as in production of the porphyrins of the instant invention for industrial purposes, where high-percentages of purity are sufficient, and medical-grade purity is not required.
The invention further comprises methods of killing microbes including Gram-positive and/or Gram-negative bacteria utilizing photodynamic porphyrin antimicrobial agents. The term microbe is used herein to include microorganisms such as bacteria, fungi, algae, and viruses. Antimicrobial agents are a family of chemical agents which kill such microorganisms or suppress their growth. An embodiment of this method comprises the steps of providing a photodynamic antimicrobial agent comprising a porphyrin with four symmetrically-distributed positive charges and a hydrophobic tail, exposing Gram-positive and/or Gram-negative bacteria to said photodynamic antimicrobial agent, and irradiating the antimicrobial agent for a period of time.
In some versions of this method, the porphyrin used is a light absorbing reduced porphyrin. In these, the reduced porphyrin may be chosen from the group of chlorin and bacteriochlorin. In other versions, the porphyrin or reduced porphyrin is a metal chelated porphyrin. In these instances, metals whose porphyrin chelates are useful as photodynamic antimicrobial agents include Mg (magnesium), Sc (scandium), Zn (zinc), Al (aluminum), In (indium), Ti (thallium), Si (silicon), Ge (germanium), Sn (tin), Pd (palladium), and Pt (platinum).
In addition, in the embodiments of the instant invention, the hydrocarbon tail may be selected from the group consisting of straight chain alkyls, straight chain alkenes, branched alkyl chains, branched alkenyl chains, aromatics, mixed alkyl-aromatic species, and mixed alkenyl-aromatic species. In some of these, the hydrocarbon tail may contain between 6 and 22 carbons (inclusive). In others, the hydrocarbon tail may contain between 6 and 18 carbons (inclusive). In still others, the hydrocarbon tail may comprise a benzyl group. In others, the hydrocarbon tail may comprise a six-carbon chain. In still others, the hydrocarbon tail may comprise a ten-carbon chain. In others, the hydrocarbon tail may comprise a fourteen-carbon chain. Further, in other embodiments, the hydrocarbon tail may comprise an eighteen-carbon chain. In preferred embodiments of the invention, white light is used in the irradiation step of the method.
In some forms of this method, the porphyrin is present in a concentration of from about 10 xcexcM to about 0.1 xcexcM. In others, the porphyrin is present in a concentration of from about 5 xcexcM to about 0.5 xcexcM. In still other forms of the embodiment, the porphyrin is present in a concentration of 1 xcexcM. In some forms of the embodiment, the period of time during which irradiation occurs may be from about 1 to about 10 minutes. In some embodiments of the invention, this period is about 5 minutes. The period of time for which irradiation is needed is determined based on the type of bacteria to be killed and the rate at which it takes up antimicrobial agent, the characteristics and concentration of the specific antimicrobial agent being used, the wavelength of the light to be used, and the intensity of the light to be used. One of skill in the art would be able to determine the period of time needed to kill or inactivate a given microbe based on these factors.
These and other objects, features, and advantages of the present invention will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter.